PROJECT SUMMARY Legionella pneumophila (Lp) causes Legionnaires' disease. Upon inhalation, Lp infects macrophages in the lung, and this is followed by severe inflammation. During infection of macrophages, Lp triggers the secretion of pro-inflammatory cytokines. Based largely upon studies done in murine models, the immune recognition of Lp is triggered by certain surface and endosomal Toll-like receptors (TLRs), cytosolic NOD-like receptors, RIG-I- like receptors, and inflammasomes. Recently, in order to discern the effect of Lp type II protein secretion on cytokine elicitation, we used shRNA to generate a panel of human macrophages that are deficient in individual immune components. While utilizing these new reagents, we made a number of observations concerning the recognition of Lp by human macrophages that deviate from / expand upon results obtained with murine cells. These findings include i) a minimal (rather than major) role for MyD88-dependent TLR signaling, ii) a larger role for the TRIF and TRAM adaptors and by extension endosomal TLR3 and/or endosomal TLR4, iii) a major role for the TBK-1 adapter, cGAS and STING, and by inference cytosolic DNA-sensing, and iv) a new role for PKR and likely other cytosolic RNA-sensors. These data call into question the field's long-standing reliance on murine models and indicate a compelling need to further define the innate immune response in human macrophage models. Also, some of our results echo findings from human epidemiological studies that have been largely ignored in recent years. Therefore, we propose to utilize knockdown protocols to discern, in the context of Lp infection of human macrophages (both U937 cell lines and those derived from peripheral blood mononuclear cells), the relative importance of TLR3 and TLR4, cytosolic DNA sensors DDX41, DNA-PK, IFI16, and MRE11, and cytosolic RNA sensors DDX3, DHX9, DHX33, DDX1/DDX21/DHX36. In order to ensure an unbiased investigation of Lp recognition by human macrophages, we will also examine knockdowns of C-type lectin receptors (Mcl, Mincle, Dectin-1, Dectin-2), which have heretofore been entirely absent from Lp studies. Examination of a parallel set of knockdowns in murine macrophages will allow us to unequivocally draw the distinction between human vs. murine infection. This line of inquiry should more readily translate into an increased understanding of and potential treatment for Lp-mediated disease in humans. Also, findings obtained with Lp have implications for other bacteria, especially intracellular pathogens of human macrophages; e.g., TLR3 and the other nucleic acid-sensing pathways have been largely studied in viral infections, and lectin receptors have been mainly examined with fungi.